Postembryonic RNAi in Heterorhabditis bacteriophora: a nematode insect parasite and host for insect pathogenic symbionts

Abstract

BackgroundHeterorhabditis bacteriophora is applied throughout the world for the biological control of insects and is an animal model to study interspecies interactions, e.g. mutualism, parasitism and vector-borne disease. H. bacteriophora nematodes are mutually associated with the insect pathogen, Photorhabdus luminescens. The developmentally arrested infective juvenile (IJ) stage nematode (vector) specifically transmits Photorhabdus luminescens bacteria (pathogen) in its gut mucosa to the haemocoel of insects (host). The nematode vector and pathogen alone are not known to cause insect disease. RNA interference is an excellent reverse genetic tool to study gene function in C. elegans, and it would be useful in H. bacteriophora to exploit the H. bacteriophora genome project, currently in progress.ResultsSoaking L1 stage H. bacteriophora with seven dsRNAs of genes whose C. elegans orthologs had severe RNAi phenotypes resulted in highly penetrant and obvious developmental and reproductive abnormalities. The efficacy of postembryonic double strand RNA interference (RNAi) was evident by abnormal gonad morphology and sterility of adult H. bacteriophora and C. elegans presumable due to defects in germ cell proliferation and gonad development. The penetrance of RNAi phenotypes in H. bacteriophora was high for five genes (87–100%; Hba-cct-2, Hba-daf-21, Hba-icd-1; Hba-nol-5, and Hba-W01G7.3) and moderate for two genes (usually 30–50%; Hba-rack-1 and Hba-arf-1). RNAi of three additional C. elegans orthologs for which RNAi phenotypes were not previously detected in C. elegans, also did not result in any apparent phenotypes in H. bacteriophora. Specific and severe reduction in transcript levels in RNAi treated L1s was determined by quantitative real-time RT-PCR. These results suggest that postembryonic RNAi by soaking is potent and specific.ConclusionAlthough RNAi is conserved in animals and plants, RNAi using long dsRNA is not. These results demonstrate that RNAi can be used effectively in H. bacteriophora and can be applied for analyses of nematode genes involved in symbiosis and parasitism. It is likely that RNAi will be an important tool for functional genomics utilizing the high quality draft H. bacteriophora genome sequence.

Cell Invasion and Matricide during Photorhabdus luminescens Transmission by Heterorhabditis bacteriophora Nematodes

“…Egg laying and IJ formation might be regulated in H. bacteriophora by a homologous pathway or by other stress response pathways (e.g., insulin-and mitogen-activated protein kinase pathways). The host biology related to symbiont transmission will likely become amenable to genetic analysis soon, as RNA interference in H. bacteriophora has recently been developed (9), and an H. bacteriophora genome project at the National Human Genome Research Institute is currently in progress (R. Wilson, personal communication).…”

“…However, better worm yields were obtained with P. temperata strain NC1 TRN16, a transmission-defective mutant that is unable to colonize the IJs and normally is associated with H. bacteriophora nematodes. An inbred strain of H. bacteriophora TT01, M31e, had been self-fertilized for 13 generations prior to use in this study (9). H. bacteriophora was maintained as a germfree stock and added to P. luminescens subsp.…”

RNAi in Agriculturally-Important Arthropods

“…For example, the symbionts Heterorhabditis bacteriophora (a nematode) and, Photorhabdus luminescens (a bacterium) are a lethal combination in some insects. Using genes described in C. elegans as a model, various phenotypes were disrupted in H. bacteriophora soaked in dsRNA (Ciche & Sternberg, 2007). These studies facilitate the discovery of genes involved in symbiosis and/or insect pathogenesis.…”

All the microbiology nematodes can teach us

“…Once the insect resources are consumed, the EPN offspring develop into the colonized infective stage and emerge to hunt for a new insect host (Herbert and Goodrich-Blair 2007;Clarke 2008). Luckily, in both types of associations, bacteria and nematodes can be cultivated independently or together, and molecular genetic techniques are available for the bacteria and, in some cases, for the nematodes (Ciche and Sternberg 2007;Goodrich-Blair 2007;Clarke 2008). This technical tractability has enabled the use of EPNs and bacteria as models of mutualism, virulence, evolution, behavior and ecology (Clarke 2008;Ram et al 2008;Adhikari et al 2009;Bode 2009;Richards and Goodrich-Blair 2009;Eleftherianos et al 2010;Hallem et al 2011;Bashey et al 2012).…”